Cancer cells, compared to normal tissue, display increased glucose uptake and metabolism via aerobic glycolysis, even in the presence of ample oxygen. This observation, termed the Warburg Effect, was paradoxical because it meant that cancer cells used a seemingly less efficient metabolic pathway to convert glucose to energy in the form of ATP. More recent investigations have demonstrated that glycolytic intermediates serve as precursors for biosynthetic pathways necessary to support cellular proliferation. The observation that cancer cells preferentially use glycolysis has led many to explore metabolism as a novel window of therapeutic opportunity for a number of tumors. Herein we propose a systematic and unbiased approach to identify glycolytic interacting proteins (GIPs) in order to better understand regulation of glycolysis in cancer. We hypothesize that glycolytic regulation in cancer cells occurs through interactions between GIPS and glycolytic enzymes and that these interactions contribute to the malignant phenotype of cancer cells. Using SILAC-based proteomics we have identified interactions enriched in cells with increased Warburg Effect, which has generated the hypothesis that increased interactions with GIPs drive glycolysis in the transformed state. We will determine which GIPs are essential for proliferation and viability using pooled RNAi screening technologies. Structural approaches will be applied to gain insights toward GIP regulatory function and this information will be used to generate tools to disrupt these potentially important interactions. Finally we will examine how transformation and the glycolytic protein interactome contribute to the overall metabolic program of the cell. The aims presented in this proposal will greatly expand our knowledge of cancer metabolism and are thus directly relevant to the mission of the National Institutes of Health.